Determination of aristolochic acids in medicinal plant and herbal product by liquid chromatography-electrospray-ion trap mass spectrometry

This paper describes a liquid chromatography-electrospray-ion trap mass spectrometry (LC-ES-ITMS) method for the determination of aristolochic acid I and II (AA-I and AA-II) in medicinal plants and Chinese herbal remedies. A reversed phase C₁₈ column with gradient elution was utilized. The effects of mobile phase additives, acetic acid and ammonium acetate, on LC separation and ES ionization were investigated. For both AA-I and AA-II, the [M+NH₄]⁺ ion was found to be the precursor ion for target MS/MS analysis. The MS/MS product ion, [M+H−44]⁺, was used for the quantitative measurement of AA-I and AA-II. The linearity was good from 0.03 to 5 μg ml⁻¹ and good correlation (r²=0.999) over the range examined was determined for both AA. The detection limit based on a signal-to-noise ratio of three was 0.012 and 0.015 μg ml⁻¹ for AA-I and AA-II, respectively. Various Chinese herbal remedies obtained from renal failure patients and medicinal plants were examined by this newly developed method.     

Determination of aristolochic acid in Chinese herbal medicine by capillary electrophoresis with laser-induced fluorescence detection

We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0 mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477 nm when excited at 405 nm using a solid-state blue laser. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0 mL MeOH, and then the extracts were subjected to centrifugation at 3,000 rpm for 5 min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples.     

Online concentration of aristolochic acid I and II in Chinese medicine preparations by micellar electrokinetic chromatography

In this study, an online concentration method in micellar electrokinetic chromatography (MEKC) applying field-enhanced sample injection (FESI) mode was developed for the detection of aristolochic acids (AAs) in Chinese medicine preparations. AA-I and AA-II were baseline separated with high separation efficiency, and 100-fold enhancement of the detection sensitivity was achieved compared with those obtained from normal capillary zone electrophoresis (CZE) or simple MEKC method. The proposed method was successfully applied for the determination of AAs in Chinese medicine preparations.     

Study on separation of aristolochic acid I and II by micellar electrokinetic capillary chromatography and competition mechanism between SDS and beta-cyclodextrin

In this study, a rapid MEKC method using 40 mM sodium borate buffer containing 50 mM SDS as surfactant was developed for the analysis of aristolochic acid (AA) in Aristolochia plants. Baseline separation of AA-I and AA-II was achieved within 3 min with high separation efficiency, satisfactory sensitivity, repeatability, and recovery. Resolution between AA-I and AA-II is above 5 and great performance with higher than 200,000 theoretical plate numbers was obtained. The detection limits (based on 3 S/N) were both 1.0 microg/mL. Two kinds of AA in 35 herbal samples of Aristolochia plants were successfully determined. The competition mechanism between beta-CD and SDS was also investigated by changing the content ratio of beta-CD and SDS.     

Simultaneous determination of five aristolochic acids and two aristololactams in Aristolochia plants by high-performance liquid chromatography

An HPLC method was developed for the simultaneous determination of five aristolochic acids (AAs) and two aristololactams (ALs) in the following six Chinese drugs derived from Aristolochia species. Samples were analyzed on a C(18) column with acetonitrile and 3.7 mm phosphoric acid buffer gradient elution, detected at 260 nm. Assay was linear over the range (microg/mL) 0.386-38.6 for aristolochic acid Va, 0.632-63.2 for aristolochic acid IVa, 0.200-20.0 for 9-hydroxy aristolochic acid I, 0.352-35.2 for aristololactam II, 0.296-29.6 for aristolochic acid II, 0.274-27.4 for aristololactam I and 3.12-312 for aristolochic acid I. Average recoveries (%) of samples were 102.0, 95.9, 99.2, 102.2, 97.2, 97.1 and 97.8 for these seven constituents, respectively. The detection limit and retention time for the seven constituents ranged from 10.0 to 15.8 ng/mL and from 12 to 21 min. As a result of drug determination, contents (in mg/g) were as follows: AA-I, 0.69-1.77; AA-II, 0.02-0.18; 9-OH AA-I, 0.04-0.12; AA-IVa, 0.76-3.36; AA-Va, 0.04-0.31; AL-I, 0.07-0.36; and AL-II, 0.01-0.09 in Madouling; AA-I, 0.03-0.41; AA-II, 0.01-0.11; 9-OH AA-I, 0.00-0.60; AA-IVa, 0.00-0.77; AA-Va, 0.00-0.14; and AL-I, 0.00-0.04 in Tianxianteng; AA-I, 1.19-4.71; and AA-II, 0.24-1.69 in Qingmuxiang; AA-I, 2.79-5.48; AA-II, 1.06-1.86; 9-OH AA-I, 0.01-0.09; AA-IVa, 0.38-0.69; AA-Va, 0.00-0.61; AL-I, 0.00-0.02; and AL-II, 0.00-0.02 in Bei-madouling-gen; AA-I, 0.64-4.23; AA-II, 0.06-0.40; and AA-IVa, 0.08-0.25; in Guangfangji; and AA-I, 1.88-9.72; AA-II, 0.26-1.88; and AA-IVa, 0.09-0.52 in Guanmutong. The other constituents were not detected in Tianxianteng, Qingmuxiang, Guangfangji and Guanmutong.     

Analysis of nephroloxic and carcinogenic aristolochic acids in Aristolochia plants by capillary electrophoresis with electrochemical detection at a carbon fiber microdisk electrode

Aristolochic acids (AAs) are the main bioactive ingredients in the most of Aristolochia plants, which are used to make dietary supplements, slimming pills and Traditional Chinese Medicines (TCMs). Excessive ingestion of AAs can lead to serious nephropathy. Therefore, quantitative analysis and quality control for the plants containing AAs is of great importance. In this paper, capillary electrophoresis (CE) with electrochemical detection (ED) at a 33 microm carbon fiber microdisk electrode (CFE) has been applied to detect AA-I and AA-II in Aristolochia plants. Under the optimum conditions: detection potential at 1.20 V, 2.0 x 10(-2) mol L(-1) phosphate buffer solution (PBS) (pH 10.0), injection time 25 s at a height of 17 cm and separation voltage at 12.5 kV, the AA-I and AA-II were baseline separated within 5 min. Low detection limits for AA-I and AA-II were 4.0 x 10(-8) mol L(-1) and 1.0 x 10(-7) mol L(-1), respectively. Wide linear ranges were from 4.0 x 10(-8) mol L(-1) to 1.9 x 10(-5) mol L(-1) and 1.0 x 10(-7) mol L(-1) to 5.0 x 10(-5) mol L(-1) for AA-I and AA-II, respectively. The proposed method has been successfully applied to analyze AAs contents in plant extracts. The results indicated that the contents of AAs in each part of Aristolochia debilis Sieb. Et Zucc. plant were different. Meanwhile, the CE-ED method was utilized for fingerprint analysis of medicine herbs. Six herbs (Radix aristolochiae, Fructus aristolochiae, Herba aristolochiae, Caulis aristolochiae manshuriensis, Caulis clematidis armandii, Caulis akebiae) were well distinguished by comparing their electropherograms obtained by CE-ED method.     

Differentiation of herbs linked to "Chinese herb nephropathy" from the liquid chromatographic determination of aristolochic acids

A HPLC method was developed and applied to analyze aristolochic acids (AA-I and AA-II) in Chinese medicinal herbs. The herb samples were extracted by using ultrasonication with the extraction efficiency of better than 82%. Extracts were then filtered and injected onto a C18 column eluting under a gradient program using methanol and water-containing 0.5% acetic acid. The method with the detection limits of 1.33 ng for AA-I and 7.29 ng for AA-II per injection was successfully applied for the analysis of traditional Chinese medicine (TCM) and related products and differentiation of Chinese medicinal herbs that have previously been misused and caused toxicological effects. The developed protocol provided an example that analysis of selected component markers could serve for health security and quality control of TCM consumption.     

葫［7］环联脲作为毛细管电泳分离介质的研究

以葫［7］环联脲(CB［7］)作为毛细管电泳添加剂,成功地分离了6种硝基苯类化合物。考察了pH值对电渗流的影响,初步评价了其基本的电泳性能,结果表明在所考察的pH值范围内(pH 2~6.5),葫［7］环联脲是质子化的,且吸附到毛细管内壁上,这使得毛细管内壁带上正电荷,电渗流反向;初步考察了葫［7］环联脲的浓度对分离的影响,证明了用葫［7］环联脲作为添加剂可完全分离对硝基乙苯、对硝基甲苯、对氯硝基苯、间二硝基苯、2,4-二硝基氯苯和硝基苯这6种物质,最佳的缓冲液为10 mmol/L Na2HPO4（用盐     

A sensitivity enhanced high-performance liquid chromatography fluorescence method for the detection of nephrotoxic and carcinogenic aristolochic acid in herbal medicines

A new, sensitive and selective HPLC method with fluorescence detector (HPLC-FLD) for the determination of nephrotoxic and carcinogenic aristolochic acid (AA) in herbal medicines by using pre-column derivatization with zinc powder in acetic acid is presented. Variables governing the derivatization reaction, such as the amount of zinc powder and acetic acid, as well as the derivatization time were studied and optimized. An extended linear dynamic range over three orders of magnitude was observed for AA-I and AA-II (R(2)>0.9998). Method accuracy at low, medium and high spiked AA levels determined by the percentage mean deviation was below 4.4% and 7.2% for AA-I and AA-II, respectively. The detection limits of 0.39 ng/mL (AA-I) and 0.52 ng/mL (AA-II) were 2 orders of magnitude lower than those obtained from HPLC-MS or CE-ECD analyses, 3-4 orders of magnitude lower than those from HPLC-UV or CE-UV methods. The developed method has been applied for the determination of AA in herbal medicines. Among the tested samples, Guanmutong had the highest AA concentration (2607.0 microg/g AA-I, 711.2 microg/g AA-II). Comparison studies between HPLC-FLD and HPLC-MS/MS demonstrated that the two methods gave similar quantitative results for the selected herb samples.     

Separation of positional isomers by cucurbit[7]uril-mediated capillary electrophoresis

A novel macrocyclic molecule, cucurbit[7]uril (CB[7]) was for the first time employed as an additive in capillary electrophoresis (CE). In similarity to other macrocyclic molecules, such as crown ethers, cyclodextrins (CDs) and calixarenes, CB[7] can form inclusion complexes with a variety of guest molecules due to its inner cavity. Thus, it can be used like other macrocyclic molecules to manipulate selectivities in CE. During the running process, CB[7] bears a positive charge under the studied pH range (pH 2.5-7) and can be adsorbed onto the inner wall of a fused-silica capillary, leading to a reversal of the electroosmotic flow (EOF). Electrophoretic behaviors of nitrotoluene, nitrophenol, nitroaniline, and methylaniline isomers were studied under various conditions. The electrophoretic separations of the isomers can be accomplished with a buffer containing CB[7]. Furthermore, a probable separation mechanism in the presence of CB[7] was also proposed.     

Determination of aristolochic acids in medicinal plants (Chinese) prepared medicine using capillary zone electrophoresis

Aristolochic acids (I and II) are commonly found in medicinal plants such as Radix aristolochiae and have been reported to cause acute hepatitis and end-stage renal failure. The aim of this work was to develop a method for the analysis of aristolochic acids in medicinal plant/Chinese prepared medicine (CPM) using (CZE). The buffer used was 30 mM sodium tetraborate at pH 9.5, detection was at 254 nm, applied voltage at 18 kV and the temperature was set at 25 degrees C. The effect of ionic strength, pH, and applied voltage on the separation was investigated. The precision values (relative standard deviation, RSD, %) for the relative migration time and peak area or peak height for aristolochic acids I and II were found to be less than 0.3% and between 2.6 to 4.0%, respectively. The limit of detection for aristolochic acids I and II was found to be 1.2 and 0.9 mg/L, respectively. The proposed method using pressurized liquid extraction (PLE) with CZE was used to determine the amount of aristolochic acids in medicinal plants or CPM samples with complex matrix and the results were compared with high-performance liquid chromatography (HPLC). Method precision (RSD, n = 6) was found to be less than 4% when those from applied to medicinal plants and CPM samples.     

Uptake,Translocation, and Fate of Carcinogenic Aristolochic Acid in Typical Vegetables in Soil−Plant Systems

When Aristolochia plants wilt and decay, aristolochic acids (AAs) are released into the soil, causing soil contamination. It has been demonstrated that aristolochic acid can be accumulated and enriched in crops through plant uptake. However, there is a lack of systematic studies on the migration and accumulation of AAs in a realistic simulated soil environment. In this study, Aristolochia herbal extracts were mixed with soil for growing three typical vegetables: lettuce, celery, and tomato. The contents of AAs in the above-mentioned plants were determined by an established highly sensitive LC-MS/MS method to study the migration and accumulation of AAs. We found that AAs in the soil can be transferred and accumulated in plants. AAs first entered the roots, which were more likely to accumulate AAs, and partially entered the above-ground parts. This further confirms that AAs can enter the food chain through plants and can have serious effects on human health. It was also shown that plants with vigorous growth and a large size absorbed AAs from the soil at a faster rate. The more AAs present in the soil, the more they accumulated in the plant.     

Examination of cucurbit[7]uril and its host-guest complexes by diffusion nuclear magnetic resonance

The self-diffusion of cucurbit[7]uril (CB[7]) and its host-guest complexes in D2O has been examined using pulsed gradient spin-echo nuclear magnetic resonance spectroscopy. CB[7] diffuses freely at a concentration of 2 mM with a diffusion coefficient (D) of 3.07 x 10(-10) m(2) s(-1). At saturation (3.7 mM), CB[7] diffuses more slowly (D = 2.82 x 10(-10) m(2) s(-1)) indicating that it partially self-associates. At concentrations between 2 and 200 mM, CsCl has no effect on the diffusion coefficient of CB[7] (1 mM). Conversely, CB[7] (2 mM) significantly affects the diffusion of 133Cs+ (1 mM), decreasing its diffusion coefficient from 1.86 to 0.83 x 10(-9) m(2) s(-1). Similar changes in the rate of diffusion of other alkali earth metal cations are observed upon the addition of CB[7]. The diffusion coefficient of 23Na+ changes from 1.26 to 0.90 x 10(-9) m(2) s(-1) and 7Li+ changes from 3.40 to 3.07 x 10(-9) m(2) s(-1). In most cases, encapsulation of a variety of inorganic and organic guests within CB[7] decreases their rates of diffusion in D2O. For instance, the diffusion coefficient of the dinuclear platinum complex trans-[[PtCl(NH3)2}2mu-dpzm](2+) (where dpzm is 4,4'-dipyrazolylmethane) decreases from 4.88 to 2.95 x 10(-10) m(2) s(-1) upon encapsulation with an equimolar concentration of CB[7].     

The formation of cucurbit[n]uril (n = 6, 7) complexes with amino compounds in aqueous formic acid studied by capillary electrophoresis

For analytes involved in dynamic equilibrium processes, capillary electrophoresis is a powerful method of determining binding constants. In this work, the complex formation between cucurbit[n]uril (CB[n] n = 6, 7) and some amino compounds was studied by capillary electrophoresis in aqueous formic acid (65% v/v). Four groups of positional and structural isomers (o, m, p-methylanilines; m, p-nitroanilines; benzidine and o-tolidine; alpha, beta-naphthylamines and 1,5-diaminonaphthalene) were selected as model compounds for study of their host-guest inclusion complexation. The interactions between CB[n] (n = 6, 7) and the model compounds were also investigated using a molecular modeling method. The results indicate that the interactions of the compounds with CB[n] (n = 6, 7) are strongly affected by the position of the substituent(s) on the aromatic ring and the ion-dipole interaction between guest molecule and CB. Furthermore, the type and the concentration of CBs on the separation and migration behavior of the amino compounds were also studied.     

Simultaneous analysis of six aristolochic acids and five aristolactams in herbal plants and their preparations by high-performance liquid chromatography-diode array detection-fluorescence detection

Aristolochic acid analogues, including aristolochic acids (AAs) and aristolactams (ALs), are known to be nephrotoxic, carcinogenic and mutagenic. In this paper, a high-performance liquid chromatography-diode array detection-fluorescence detection (HPLC-DAD-FLD) method was developed for the simultaneous determination of six AAs together with five ALs. Baseline separation was obtained on an ODS C18 analytical column with 0.2% HAc/methanol gradient elution. The hyphenation of DAD and FLD allows the method to directly meet the analysis requirements of most herbal plants with high sensitivity and selectivity. For trace analysis, aristolochic acids were reduced to their corresponding aritstolactams in acidic solution containing iron powder, and then high sensitive detection and quantification were carried out. The method was successfully validated in the matrices of various Aristolochiaceae plants and their preparations. Linearities of around 3-4 orders of magnitude were obtained with correlation coefficients exceeding 0.9970. The detection limits were decreased to 0.2ng/ml. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 5.74%, and the average recovery factors were in the range of 94.5-99.2%.     

硅藻土载负葫芦[7]脲气相色谱固定相的制备及其性能评价

在酸性条件下,将自制的葫芦[7]脲均匀地涂覆到102白色硅藻土担体上,制得葫芦[7]脲气相色谱固定相。 采用红外光谱、质谱、元素分析和热重分析表征了葫芦[7]脲在载体表面的结构。 利用相关探针测定了新固定相的麦氏常数,表征了其基本色谱性能。 考察了葫芦[7]脲气相色谱固定相填充柱对芳香烃、卤代烃、醇、酮、酯及硅氧烷的分离能力。 结果表明,葫芦[7]脲固定相热稳定性高,柱色谱性能稳定,对较广泛的化合物尤其对高沸点的酯类及硅氧烷类化合物显示出良好的色谱分离能力(7 min内分离),作为气相色谱固定相有较好的应用前景。 初步讨论了葫芦[7]脲固定相对上述化合物的分离机理。     

Organic and inorganic di-cations for capillary silica coating and EOF modulation in CE: Example of application in PEG analysis

EOF measurements, by using 1,4-di-(4-aza-1-azonia-bicyclo[2.2.2]octane)butane diiodide, barium and strontium tetraborate as silica wall modifiers, are reported and, as an example of application, analysis of PEG (PEG 400-2000) polydisperse preparations in free solution CZE is shown. PEGs have been derivatized with phthalic anhydride so as to form singly or doubly charged derivatives with strong UV absorbance at 214 nm. Whereas separations in plain tetraborate buffer, pH 9.0, without any EOF control, did not lead to good resolution of all-size oligomers and suffered from long analysis times, excellent resolution of all oligomers up to 40 ethylene oxide (EO) units could be obtained under EOF control. Such EOF modulation was engendered by addition of 1 mM M7C4M7, a doubly charged organic cation able to stick tenaciously to the silica wall. Further modulation of EOF and silica surface modification could be achieved also by addition of inorganic cations, notably those of group II, whereas monovalent cations did not seem to affect much the EOF flux. Among the doubly charged cations investigated, Ca++, Mg++, Sr++ and Ba++, the latter did seem to offer best EOF control and reproducible runs. A judicious blend of M7C4M7 (0.33-1 mM range) with barium (10-20 mM range) allowed baseline resolution of all PEG oligomers investigated up to PEG 2000 and >40 EO units in length. In this last case, best results in terms of reproducibility and separation efficiency of the more heavy homologues were obtained using Li+ salt in small amounts.     

On-column complexation capillary electrophoretic separation of Fe2+ and Fe3+ using 2,6-pyridinedicarboxylic acid coupled with large-volume sample stacking

On-column complexation of Fe2+ and Fe3+ with 2,6-pyridinedicarboxylic acid (2,6-PDCA) formed anionic complexes, which were then separated by capillary zone electrophoresis with direct UV detection at 214 nm. To achieve reasonable separation selectivity and on-column complexation, the conditions such as pH, the concentration of 2,6-PCDA and the EOF modifiers in the electrolyte were examined. The electrolyte contained 5.0 mM 2,6-PDCA, 0.25 mM tetradecyltrimethlammonium bromide (TTAB) and 5% (v/v) acetonitrile at pH 4.0 was optimised for on-column complexation and the separation of Fe[PCDA]2(2-) and Fe[PCDA]2(-). To enhance the detection sensitivity, large-volume sample stacking (LVSS) was used for the on-line preconcentration of Fe[PCDA]2(2-) and Fe[PCDA]2(-). Under the optimised conditions, satisfactory working ranges (0.5-50 microM), lower detection limits (less than 0.1 microM) and good repeatability of the peak areas (R.S.D.: 5.2-7.8%, n = 5) was achieved using LVSS (300 s). With LVSS, the detection sensitivity was enhanced more than 50-fold compared to conventional hydrodynamic injection. The proposed method was used successfully for the determination of Fe2+ and Fe3+ in water samples.     

Pressurized liquid extraction of berberine and aristolochic acids in medicinal plants

Berberine and aristolochic acids I and II present naturally in medicinal plants were extracted using a laboratory-made pressurized liquid extraction (PLE) system in the dynamic mode. As the target analytes were present naturally in the medicinal plants, spiking was not done and comparison with ultrasonic extraction and Soxhlet extraction was performed to assess the method accuracy. The effect of temperature, volume of solvent required and particle size were investigated. Method precision (RSD, n=5) between 1.98 and 3.4% was achieved for the extraction of berberine and aristolochic acids I and II in medicinal plants and lower than 8% for lower levels of aristolochic acid II in medicinal plants.     

Dynamically coated silica monolith with ionic liquids for capillary electrochromatography

An ionic liquid, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]) was introduced as dynamic coating of a silica monolithic column for capillary electrochromatography of phenols and nucleoside monophosphates. The run-to-run and column-to-column repeatability of migration time for six phenols were satisfactory on this column with relative standard deviation values less than 0.90 and 4.31%, respectively. Anodic electroosmotic flow (EOF) was observed, which increased with the increase of [BMIM][BF4] concentration within 120 mM and when [BMIM][BF4] concentration was above 120 mM, EOF leveled off due to the saturation of [BMIM][BF4] on the monolith. Efficient separation of phenols and nucleoside monophosphates on this dynamically coated monolithic column was obtained, compared with a dynamically coated fused-silica column and unmodified silica monolithic column. The retention behavior of uncharged phenols is mainly manipulated by hydrophobic interactions due to the presence of butyl groups, and that of nucleoside monophosphates is governed by the electrostatic attraction mechanism based on the interaction between positively charged [BMIM][BF4] moieties and negatively charged phosphate groups. In addition, silica matrix also contributes to the separation resolution.